This invention relates to video imaging systems.
Basic research in cell biology and physiology is interested in isolating the different chemical participant molecules and understanding the working relationship between them.
For example, the role of calcium as a second messenger in a variety of cell responses is a prime target for this kind of study. A large calcium concentration gradient is preserved by pumps transporting the free Ca.sup.++ ions out of the cell, or into calcium stores inside the cell. Due to the large concentration gradient, intracellular calcium levels can rise from 100 nM to above 1 .mu.M in a matter of a few seconds. Therefore, quantitative spatial information that is minimally corrupted by motion artifacts or noise is necessary to study changes in Calcium concentration.
Fluorescent dye agents can be used to quantify the presence of distinct molecules within a population of whole cells or in isolated cell compartments. There is a wide availability of fluorescent tracers for specific proteins, lipids, and ions; as well as stains for different cell structures, and probes that change spectral properties with pH, or membrane potential.
Since cell responses to external stimuli may vary from the millisecond range to several hours, a sensitive digital imaging microscope together with the appropriate fluorescent labels permit an investigator to follow the target molecules spatially within the cell.
In principle, any two dyes that do not overlap in their excitation and emission spectra can be imaged in rapid succession. Ratiometric indicators, that require fluorescent distribution data at two different wavelengths, can provide reliable quantitative data.
Only a fraction of the absorbed photons will actually promote the emission of fluorescence photon. This fraction reflects any alternate ways of de-energizing and is dependent upon the fluorescent species and the excitation wavelength. This fraction is called the Quantum Efficiency (QE) and is always less than 1. This fraction is given by the relationship: Quantum Efficiency=Photons emitted/Photons absorbed So, the rate of fluorescent photon emission (F) is given by the equations: EQU F=QE *Ka
where K.sub.a =I.sub.o -I and is the rate at which photons are absorbed.
This can be rewritten as: EQU F=QE * I.sub.o * (1-exp(-( e*c*L )))
where (C) is the concentration of the molecules, (L) is the path length, (e) is the extinction coefficient and I.sub.o is the incident light intensity.
Using the series expansion equivalent for the exponential function this equation can be expanded: EQU Exp (X)=1+X+X.sup.2 /2!+X.sup.3 /3!+. . .
For low concentrations (c) and short path lengths (L), the fluorescent intensity equation can be approximated by: EQU F.apprxeq.QE * I.sub.o * e * c * L
It can be seen that the fluorescent intensity in photons per seconds is directly proportional to incident intensity, extinction coefficient, quantum efficiency, concentration, and pathlength.
With the advances in biochemistry over the last decade several fluorescent probes have been developed to study cells. Probes can be used to covalently label macromolecules or organelles in living and fixed cells. DNA, RNA, proteins and lipids can be labelled. Immunochemistry assays can produce fluorescently labelled antibodies that bind with a high affinity to specific protein receptors or enzymes. There is also another group of fluorescent probes that will change their fluorescent intensity o spectra following changes in specific ion concentrations, pH, membrane potential, etc.
It is thus now possible to study cell function by correlating the distributions of different molecules or ions simultaneously in single living cells. By labelling specific organelles, it is also possible to determine the redistribution of target molecules or ions after an experimental stimulus is applied. Local concentrations of target molecules or ions can be calculated by using probes that alter their fluorescent response in the presence of these chemical species.
The precise determination of local concentrations from fluorescent data in single cells is difficult due to the low fluorescence intensity that can be obtained from single cells. Even with the calibration curves that associate the intensity value detected to local concentration values, there are several sources of error. Changes in optical pathlength (thickness) in different regions of the cell, and any preferential distribution of the probe in different cell compartments will undermine the calculations.
Ratiometric indicators, that shift their spectral peaks upon binding ions such as calcium or sodium are now commercially available. With the use of these indicators, the local concentration of the ion can be calculated from data acquired at two different wavelengths.
Consider an ideal fluorescent probe that has only two possible configurations:
1) Bound to the ion target: With spectral peak at wavelength L.sub.1 PA1 2) Unbound (free): With spectral peak at L.sub.2 PA1 a.sup.f.sub.1 / a.sup.f.sub.2 =F.sup.f.sub.1 / F.sup.f.sub.2 =ratio of fluorescence for the free form of the dye, PA1 a.sup.b.sub.1 / a.sub.2 =F.sup.b.sub.1 / F.sup.b.sub.2 =ratio of fluorescence for the bound form of the dye, and PA1 a.sup.f.sub.2 / a.sup.b.sub.2 =F.sup.f.sub.2 / F.sup.b.sub.2 =ratio of fluorescence at wavelength 2. PA1 R=F.sub.340 /F.sub.380 =Ratio of fluorescence intensity in cell, PA1 Rmin=F.sup.f.sub.340 /F.sup.f.sub.380 =Ratio of fluorescence for free form of the dye, PA1 Rmax=F.sup.b.sub.340 /F.sup.b.sub.380 =Ratio of fluorescence for Calcium bound form of the dye, PA1 .beta.=F.sup.f.sub.380 /F.sup.b.sub.380 =Ratio of fluorescence at 380 nm, and PA1 Kd=Dissociation constant for Fura-2, which is approximately 220 nM in vitro.
To make use of the largest shift in spectra, two wavelength measurements (F.sub.1 and F.sub.2) are made, corresponding to the spectral peaks, L.sub.1 and L.sub.2. It is important to note that the spectral curve of these dyes is usually very wide, and there is some overlap between the free and the bound species curves.
Since only two possible fluorescent states exist, any measurement of fluorescent intensity (F.sub.1 and F.sub.2) of a mixed solution (or loaded cell) will be the additive contributions from the fluorescence of the two species.
The fluorescence at wavelength 1 that is due to the free (f) and the bound (b) form of probe is given by the expression: F.sub.1 =F.sup.f.sub.1 +F.sup.b.sub.1
Similarly, the fluorescence at wavelength 2 is given by the expression F.sub.2 =F.sup.f.sub.2 +F.sup.b.sub.2
The unknown partial contributions: F.sup.f.sub.1, F.sup.f.sub.2, F.sup.b.sub.1, and F.sup.b.sub.2 are a function of excitation intensity (I.sub.o), pathlength (L), concentration (c), quantum efficiency (QE), and extinction coefficient (e).
Letting a=QE * e,
And c.sup.f, c.sup.b =concentration of free and bound form of the probe respectively, then EQU F.sub.1 ={( a.sup.f.sub.1 * c.sup.f )+( a.sup.b.sub.1 * c.sup.b ) }* I.sub.o * L EQU F.sub.2 ={( a.sup.f.sub.2 * c.sup.f )+( a.sup.b.sub.2 * c.sup.b ) }* I.sub.o * L
The chemical dissociation equation for the dye is: EQU c.sup.b .fwdarw.c.sup.f +[ION]
Hence, EQU Kd=c.sup.f * [ION]/ c.sup.b
If the constant of dissociation (Kd) of the probe-ion binding equation is known, then the actual ion concentration ( [ION] ) is found by substituting this last equation as into the two previous equations before taking their ratio. ##EQU1##
From the above equation it is seen that the ratio of fluorescence is independent of pathlength, intensity, and concentration of probe. The ratio is only a function of ion concentration ( [ION] ), which can be now calculated without regard to cell shape. ##EQU2##
When calculating ion concentration from acquired data it is not necessary to consider any of these intermediate parameters constants, since it can be found from the ratio of fluorescence intensities. That is:
Kd is the constant of dissociation and can be measured in solutions in vitro.
Another consideration is that the fluorescent dyes used to label living cells have to be introduced into the cell cytoplasm with a minimum of damage to cell function.
Some dyes can be made membrane soluble (non polar) by adding chemical species to their polar ends. The cells are then submerged in media containing the membrane soluble form of the dye for loading. After the dye diffuses from the media in which it is barely soluble into the cell membrane, enzymes inside the cell cleave the terminal species away leaving the fluorophor trapped inside the cell. This method provides the least disruptive approach to cell loading and has a greater cell survival rate.
For example, Fura-2 dye can be obtained in a free acid form and in the acetoxymethyl (AM) form. The free form is calcium-sensitive and not membrane-soluble, while the Fura-2 AM form can move into the membrane but does not respond to changes in calcium concentration. Intracellular esterases hydrolyze the AM form into the free acid form. The cells must not be overloaded with Fura-2 AM dye, as this will overwhelm the esterase capacity of the cell, and result in incomplete cleavage of dye which will affect the fluorescent measurements, since this intermediate form is highly fluorescent, but insensitive to local calcium concentration.
Fura-2 dye shifts the peak excitation from 380 nm to 340 nm when it binds to calcium. The equation for calcium concentration ([CA.sup.++ ]) requires the values for fluorescence intensity at the two wavelengths for both the calcium bound and the calcium free form of the dye. That is: EQU [Ca.sup.++ ]={(R-Rmin)/(Rmax-R)}* Kd * .beta.,
where
To obtain the values for the constants: Rmin, Rmax, and .beta., fluorescent solutions with a known calcium and Fura-2 concentrations are imaged. The calcium free solution is made by adding the Ca.sup.++ chelator EGTA to the calibration solution, and Fura-2 free acid to a concentration of 1 to 4 micromolar. The calcium bound solution includes the sustaining media with extra calcium and the Fura-2 free acid. With these values, the grey levels obtained from the individual video images at each wavelength can be related to calcium concentrations inside the cell. Rmin, Rmax, and .beta. should be determined under the same chemical conditions (temperature, pH, concentration) and with the same optical components (objective, filters, and dichroic mirror) as the recorded images.
Commercial video cameras of the vidicon type require a photon flux greater than 10.sup.8 photons per millimeter squared per second to provide an "acceptable" (10.1 Signal to noise) image. Such cameras are generally too insensitive for detecting fluorescent images.
With the advent of the first generation image intensifiers in the 1960s, researchers use of intensified video cameras in microscopes for biological research. In the early 1970s, the Silicon Intensified Camera (SIT camera) pushed the sensitivity frontier to 10.sup.7 photons/mm2/sec. High quality intensified cameras and video recorders promoted a wider use among biological researchers. Second generation intensifiers, smaller and with simpler support electronics can be used as a first stage for a SIT camera (called an ISIT), with a sensitivity of 10.sup.5 photons/mm2/sec. SIT and ISIT cameras are used routinely to image fluorescent probes in living cells, but have problems of image persistence and geometrical distortion. Parallel advances in solid state technology in this decade have provided sensors like the charge coupled device (CCD), and charge injection device (CID) without the problems mentioned above. However, these sensors require an intensification stage to be used in low light level imaging at video rates (30 frames/second).
The development of the imaging technology was closely followed by biological applications. The fluorescent protein aequorin, which fluoresces when exposed to micromolar concentrations of calcium, was one of the first labels to be used in low light level microscopy. Experiments using aequorin showed that most of the calcium present in the cell in not in the free ion state, but sequestered, or stored inside. A sequence of images has been obtained which showed a "wave" of calcium spreading through the plasma membrane of a medaka fish egg upon fertilization. Other common fluorescent probes including "Rhodamine", "Texas Red", and "Fluorescein", have been attached to antibodies for proteins, lipids and other macromolecules. Many researchers have studied cell function, structure and vitality using the newly available instrumentation and fluorescent probes. Most of these studies required the averaging of several video frames to obtain an image with a good signal to noise ratio (SNR), and the processes being studied were not fast enough that this averaging would pose a problem.